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Biochimica et Biophysica Acta 1758 (2006) 1053–1060 www.elsevier.com/locate/bbamem

Review -5 water channel in lipid rafts of rat parotid glands ⁎ Yasuko Ishikawa a, , Gota Cho b, Zhenfang Yuan c, Noriko Inoue d, Yoshiko Nakae e

a Department of Medical Pharmacology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan b Dental Anesthesiology, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan c Department of Internal Medicine, Peking University First Hospital, Xishiku Street, Xicheng District, Beijing 100034, China d Department of Oral Care and Clinical Education, Tokushima University Medical and Dental Hospital, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan e Oral and Maxillofacial Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan Received 18 October 2005; received in revised form 2 March 2006; accepted 21 March 2006 Available online 19 April 2006

Abstract

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or α1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or α1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence. © 2006 Elsevier B.V. All rights reserved.

Keywords: Aquaporin-5; Lipid rafts; Parotid glands; Diabetes; Aging; Interlobular ducts

Contents

1. Introduction...... 1053 2. Distribution of AQP5 in salivary gland cells ...... 1054 3. Association of AQP5 with lipid rafts in rat parotid gland cells ...... 1054 4. Translocation of AQP5 together with lipid rafts to the APM and its dissociation to non-rafts in rat parotid gland cells...... 1055 5. Water movements and the osmolarity generation in parotid interlobular ducts ...... 1056 6. Mechanisms underlying the translocation of AQP5 towards the APM ...... 1056 7. The mechanisms underlying age- and diabetes-related impairment of the responsiveness of AQP5 to cholinergic stimulation . . . 1057 8. Conclusion ...... 1058 Acknowledgements ...... 1059 References ...... 1059

1. Introduction such as amylase and mucin from the secretory granules in the acinar cells by exocytosis due to activation of β2- Rat parotid glands are innervated by both the sympathetic adrenoceptors. Noradrenalin (Nor) released by sympathetic and parasympathetic nerves of the autonomic nervous system. stimulation also activates α1-adrenoceptors on the cells and Sympathetic stimulation induces the secretion of salivary induces modest salivary fluid secretion [1]. Parasympathetic stimulation induces the largest salivary fluid secretion as a ⁎ Corresponding author. Tel.: +81 88 633 7332; fax: +81 88 633 7332. result of the activation of M3 muscarinic acetylcholine (ACh) E-mail address: [email protected] (Y. Ishikawa). receptors (mAChRs) on the cells [1]. The main component of

0005-2736/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbamem.2006.03.026 1054 Y. Ishikawa et al. / Biochimica et Biophysica Acta 1758 (2006) 1053–1060 saliva is water, and the molecular mechanisms by which water of salivary fluid secretion is accompanied by the translocation is secreted from the salivary gland cells remain unknown, of AQP5 together with lipid rafts into the APM via intracellular because of the presence of the plasma membranes as a major Ca2+-signaling and dissociation of AQP5 from the lipid rafts at barrier. the APM, and the molecular mechanisms that underlie impaired In 1988, a 28-kDa integral membrane , now known as responsiveness of AQP5 to neurotransmitters as a consequence aquaporin-1 (AQP1), was discovered as a water channel [2,3]. of aging or diabetes-related xerostomia. There have been many attempts at homology cloning to isolate water channel proteins and to identify sequence-related 2. Distribution of AQP5 in salivary gland cells proteins in many kinds of living cells. AQP water channel proteins are abundant in a variety of cells for the transportation AQP5 has been identified with AQP1, AQP3, and AQP8 in of fluid and have homology with the of mammalian salivary glands [26]. AQP1 is expressed in the cap- the lens, which form water channels [4]. Thirteen members of illary endothelial cells of rat parotid and submandibular glands [27]. the AQP family, AQP0–AQP12, have been identified from AQP8isalsoexpressedinratsalivaryglands[28,29],butthe many mammalian cells [5,6]. The AQP family consists of three localization of it is not clear. AQP5 is localized in the APM of subsets coresponding to AQPs, aquaglyceroporins, and super- serous acinar cells of rat submandibular [14,20] and parotid [15– . AQPs selectively permeate water and consist of 17,21,23–26] glands, and of interlobular duct cells of rat parotid AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and AQP8. glands [16]. AQP5 is also localized in the intracellular structures in Aquaglyceroporins permeate water and glycerol and consist of rat parotid gland cells [15,16,30,31]. To directly visualize the AQP3, AQP7, AQP9, and AQP10 [5]. Superaquaporins have subcellular localization of AQP5 in rat parotid glands, a histologic poorly conserved asparagin–proline–alanine (NPA) boxes and approach was used [16]. Confocal immunofluorescence micro- consist of AQP11 and AQP12 [6]. scope images revealed that in the interlobular duct cells of rat Recently, studies have focused on the mechanisms underly- parotid glands under resting conditions, AQP5 is located in the ing the control of AQPs to clarify the molecular basis of water intracellular structuresaswellasintheAPM(Fig. 1) [16].To movement across biologic membranes in relation to disease [4– quantify the subcellular distribution of AQP5, the interlobular duct 13]. Impaired AQP2 trafficking causes nephrogenic diabetes cells were analyzed in sections from control rat parotid glands with insipidus [8,9]. Changes in AQP1, AQP4, and AQP9 expression immunoelectron microscopy. Immunogold labeling at the APM in the brain are correlated with edema formation [10,11]. and within the intracellular compartment was assessed on sections Autoantibodies on the mAChRs in Sjogren's syndrome cause to from unstimulated parotid glands of different rats. Under resting decrease pilocarpine-induced AQP5 trafficking to APM of HSG conditions, 90% of the immunogold particles were associated with cells [13], suggesting the importance of AQP5 trafficking to the intracellular compartments, the remaining 10% were associated APM of salivary glands in mACh agonist-induced exocytosis. with the APM. Thus, under resting conditions, AQP5 was mainly The AQP5 cDNA, which encodes a 265-residue polypeptide, localized in intracellular vesicular structures in the interlobular duct was isolated from rat submandibular gland with the use of a cells of rat parotid glands. AQP5 is located in the cytoplasmic homology-based cloning approach by Raina et al. [14]. AQP5 is vesicles [30] and the secretory granule membranes [31] of rat highly expressed in the apical domains of rat serous acinar cells of parotid glands. AQP1 is localized in the zymogen granules in rat exocrine glands, such as the parotid and submandibular glands exocrine pancreas [32]. AQP6 is located in the intracellular vesicles [15–17], lacrimal glands [18], and sweat glands [19], subepithe- in renal epithelia [33]. Thus, different AQP homologues are lial glands of the upper airway [20], and rat interlobular duct cells localized in different cell membranes and in various intracellular of parotid glands [16]. AQP5 is predicted to have a significant organelles in many kinds of cells. role in fluid secretion from serous acinar cells of salivary glands on the basis of its abundance in the apical region of the cells. This 3. Association of AQP5 with lipid rafts in rat parotid gland is supported by the fact that knockout mice lacking AQP5 have cells markedly decreased rates of salivary secretion [21]. Activation of M3 mAChRs and α1-adrenoceptors on the Under resting conditions, AQP5 is localized in the intra- acinar cells of rat parotid glands increase the intracellular cellular structures of rat parotid gland cells (Fig. 1) [15,16]. The 2+ 2+ concentration of Ca ([Ca ]i) and induces salivary secretion intracellular structures where AQP5 is located in rat parotid from the cells [1,22–25], indicating the importance of Ca2+- gland cells remain to be characterized. Cholesterol- and glyco- mediated intracellular signal transduction for the secretion. sphingolipid-enriched microdomains, commonly known as lipid Whether the subcellular distribution of AQP5 is regulated by rafts, were recently suggested to be involved in a number of cell Ca2+-signaling through M3 mAChRs and α1-adrenoceptors in functions, such as membrane sorting and trafficking [34–39], parotid glands, however, was unknown. We studied whether the receptor signaling [40], and cholesterol homeostasis [41]. For stimulation of salivary fluid secretion is accompanied by the example, transcytosis of IgA and exocytosis of newly made redistribution of AQP5 into the apical plasma membranes brush-border proteins in enterocytes occur through an apical (APM) from the intracellular structures, the regulatory mechan- lipid raft-containing compartment [42]. These findings suggest isms of the redistribution of AQP5, and the characterization of that lipid rafts are involved in sorting some apical resident the structures where AQP5 is located in the parotid gland cells proteins. In order to characterized the intracellular vesicles in rat [13–17,21,23–26]. We also focused here whether the induction parotid glands where AQP5 is located, flotillin-2 and GM1 Y. Ishikawa et al. / Biochimica et Biophysica Acta 1758 (2006) 1053–1060 1055 ganglioside, which are a lipid raft-associated integral membrane localization of AQP5 in lipid rafts [16]. These results indicated protein and glycosphingolipid, respectively, were used as raft that AQP5 is located in lipid rafts in the cytoplasm of parotid markers [38] to determine if AQP5 is located in lipid rafts in rat gland cells under resting conditions. parotid gland cells. Confocal immunofluorescence microscope images revealed that under resting conditions, AQP5 was colo- 4. Translocation of AQP5 together with lipid rafts to the calized with flotillin-2 and GM1 in the cytoplasm of rat parotid APM and its dissociation to non-rafts in rat parotid gland gland cells, indicating that AQP5 is located in lipid rafts [16]. cells This result was further supported by sucrose density gradient experiments in which AQP5 in rat parotid gland tissues, under It is well documented that salivary fluid secretion is induced unstimulated conditions, fractionated to the same fractions con- by the binding of neurotransmitters from autonomic nerve taining flotillin-2 and GM1 [16]. Lipid rafts migrate to the lighter endingstoM3mAChRsorα1-adrenoceptors localized on the density fractions in an Opti-Prep discontinuous density gradient basolateral plasma membrane of parotid acinar cells [1].The [39,43]. After separation of the rat parotid tissue homogenate on responsiveness of AQP5 in salivary gland cells to stimulation the Opti-Prep gradient, the amount of AQP5 in the tissues under with M3 agonists or α1 agonists was investigated to evaluate the resting conditions was highest in the lighter fractions, indicating role of AQP5 in salivary secretion. Treatment of the tissues with that AQP5 is located with lipid rafts in resting parotid gland cells ACh induces AQP5 translocation from the intracellular of rats. Next, to test the solubility of AQP5 in cold detergent, rat structures to the APM within 1 min, but treatment for more parotid slices were treated with solution containing 1% Triton X- than5minresultsinAQP5translocationfromtheAPMtothe 100 for 30 min at 4 °C [35,39]. The homogenate was then intracellular structures [15]. SNI-2011, cevimeline, acting at M3 centrifuged to separate TritonX-100-insoluble and -soluble receptors induces a long-lasting increase in the amount of AQP5 fractions. Under resting conditions, the major portion of AQP5 in the APM of rat parotid glands [24]. Treatment of rat parotid was present in the Triton X-100-insoluble fraction, revealing the tissues with epinephrine also induces transient and marked

Fig. 1. Changes in confocal immunofluorescence microscope images of AQP5 and CLC-K2 in interlobular duct cells and acinar cells of parotid glands of rat injected with cevimeline. Parotid glands were obtained from rats injected with physiologic saline (A and D) and cevimeline (5.0 mg/kg) (B, C, E and F). The parotid glands were removed and embedded 0 min (A), 10 min (B and E), or 60 min (C and F) after injection. The section was immunostained to detect AQP5 using Alexa-488 (green, A, B, and C) and Alexa-568 (red, D-2, E-2, F-2) or CLC-K2 using Alexa-488 (green, D-1, E-1, and F-1). Nuclei were stained with ethidium bromide. Scale bars: 10 μm. 1056 Y. Ishikawa et al. / Biochimica et Biophysica Acta 1758 (2006) 1053–1060 trafficking of AQP5 between intracellular structures and the AQP5 as shown in Fig. 1E-1 and E-2 [61],suggestingthatCLC- APM [23]. Cytochalasin D and tubulozole-C inhibited the K2 play a role to make osmotic gradients. Further experiments translocation of AQP5 to the APM, suggesting the importance are, however, necessary to clarify the interaction Cl− channel with of the cytoskeleton in this translocation [22,44]. The trafficking the appearance of the function of AQP5 in salivary glands. of AQP5 induced by cholinergic or adrenergic stimulation in rat In the first stage, saliva is secreted initially from the acinar cells parotid glands was directly visualized with immunohistochem- [50]. This saliva is called primary saliva and is plasma-like in + − − istry [16]. Immunoelectron microscopy of AQP5 expression in concentration of Na ,Cl , and HCO3 . In the second stage, the interlobular duct cells of rat parotid gland revealed that 10 min primary saliva is modified in duct cells. It was notified by the old after the intravenous injection of cevimeline, the percentage of theory that the ducts seem to be impermeability to water, but Na+ gold particles in the APM rose to 70% from 10% (under resting is actively reabsorbed and K+ secreted from the duct cells [50]. conditions) and that of the remaining gold particles localized However, there are ample evidences for permeability to water in intracellularly fell to 30% from 90% (under resting conditions), parotid interlobular ducts. For example, forskolin or carbachol indicating the translocation of AQP5 to the APM in response to evokes water secretion from rat parotid interlobular duct, asso- cevimeline treatment. Confocal immunofluorescence microscope ciated with secretion through CFTR and diphenylamine-2- images revealed that cevimeline induced the translocation of the carboxylate-sensitive anion channels of carbonic anhydrase- immunofluorescence of AQP5 together with that of flotillin-2 or dependent bicarbonate linked with the Na+/H+ exchange GM1 towards the APM in rat interlobular duct cells 10 min after mechanism [51,52], suggesting that salivary fluid is secreted intravenous injection into the tail vein (Fig. 1B) [16].Sixty from parotid interlobular duct cells as well as acinar cells. minutes after the treatment, immunofluorescence was dispersed throughout the cell (Fig. 1C). These results indicate that AQP5 6. Mechanisms underlying the translocation of AQP5 translocates with lipid rafts to the APM of interlobular duct cells towards the APM of rat parotid glands, suggesting that water is secreted from interlobular duct cells of rat parotid glands as well as acinar Salivary fluid secretion is controlled through M3 mAChRs and cells. This suggestion is also supported by several cell lines of α1-adrenoceptors [1]. AQP5 is likely to be a target molecule for evidence of an important role for the ductal system in fluid the control of saliva production. The treatment of rat parotid secretion in pancreas [45]. tissues with ACh, cevimeline, or epinephrine induces the As mentioned above, the major portion of AQP5 was present translocation of AQP5 between the APM and the intracellular in the TritonX-100-insoluble fraction under resting conditions of structures [15,16,23,24]. The translocation of AQP5 induced by rat parotid gland tissues. Conversely, after the treatment with M3 mAChR agonists is inhibited by an inhibitor of calcium cevimeline, the amount of AQP5 decreased in the TritonX-100- release from the intracellular compartment, TMB-8, but not by insoluble fraction and increased in the TritonX-100-soluble frac- protein kinase C (PKC) inhibitors, H-7 and GF 109203X. Con- tion. In contrast, cevimeline did not change the amount of flotillin- versely, a PKC activator, phorbol 12-myristate 13-acetate, does 2 and GM1 in the Triton-X-100-insoluble fraction. These results not induce the translocation of AQP5 [24]. The calcium indicate that M3 mAChR agonists and α1-adrenoceptor agonists ionophore A-23187 induces the translocation of AQP5 between induces the translocation of AQP5 with lipid rafts from the intra- the APM and the intracellular structures [15,16]. Epinephrine cellular structures to the APM and the dissociation of AQP5 from acting at α1-adrenoceptors in parotid glands also induced the lipid rafts to non-rafts at the APM in rat parotid glands [16], translocation of AQP5 from the intracellular structures to suggesting that the lipid-AQP5 interactions are important to play the APM [23]. The translocation of AQP5 induced by ACh the function of AQP5 on the APM. or epinephrine is inhibited by a phospholipase C inhibitor, U73122, as well as the inhibitors of calcium release inhibition 5. Water movements and the osmolarity generation in from intracellular stores, dantrolene, and TMB-8 [24].Fluores- 2+ parotid interlobular ducts cence studies with fura-2/AM demonstrate that [Ca ]i rapidly increases in a concentration-dependent manner with marked Water moves passively in response to osmotic gradients fluctuations after exposure of the isolated parotid acinar cells to − − generated by active Cl driven HCO3 secretion [46]. Salivary ACh and cevimeline [24,25], and that the elevation of AQP5 − 2+ 2+ gland express multitype types of Cl channels including Ca - levels in the APM coincided with the elevation of [Ca ]i.The dependent and volume-sensitive channels [47], the cystic fibrosis presence of extracellular Ca2+ was necessary for the maximum transmembrane conductance regulator (CFTR)[48] and the effect of ACh on the increase of AQP5 in the APM in parotid voltage-regulated ClC-2 and ClC-3 Cl− channels [49].CFTRis tissues [25]. An inhibitor of myosin light chain kinase (MLCK), located in interlobular ducts of rat parotid glands [48] and ML-9, inhibited ACh- or pilocarpine-induced increases in the cholangiocytes of rat liver [46]. Secretin and dibutyryl-cAMP AQP5 levels in the APM [25]. MLCK was identified in parotid stimulate the translocation of CFTR from intracellular vesicles to glands and regulates capacitative Ca2+ entry [53].Itissuggested the APM [46]. Recently, we found that CLC-K2 (Cl− channel) that both the Ca2+ release from intracellular stores and the entry of was also expressed in the cytoplasm of interlobular duct cells of Ca2+ into cells regulate the translocation of AQP5 from the rat parotid glands and colocalized with AQP5 in lipid rafts under intracellular structures to the APM in rat parotid glands. unstimulated conditions (Fig. 1D-1) [61]. Ten minutes after the Furthermore, to confirm the involvement of Ca2+ in the trans- injection of cevimeline, CLC-K2 moved to APM together with location of AQP5, the Ca2+ ionophore, A-23187, was injected Y. Ishikawa et al. / Biochimica et Biophysica Acta 1758 (2006) 1053–1060 1057 intravenously into rat tail vein [16]. Ten minutes after the in- respectively, block NO donor-induced increases in AQP5 in the jection, AQP5 fluorescence was predominantly associated with APM. KN-62, an inhibitor of CaM kinase ∣∣, decreases the the APM of the interlobular duct cells of parotid glands. Treat- pilocarpine-induced increase in AQP5 in the APM [25]. A study ment of the parotid tissues with A-23187 decreased the amount of using diaminofluorescein-2 diacetate demonstrates enhanced AQP5 in the TritonX-100-insoluble fraction [16]. These findings NOS activity in isolated parotid acinar cells in real time after 2+ suggest that increases in [Ca ]i mediate the effects of cevimeline ACh-treatment. Treatment with dibutyryl cGMP, but not dibu- on the movement of AQP5 with lipid rafts from the cell cytoplasm tyryl cAMP, induces an increase in AQP5 in the APM. BAPTA- to the APM and subsequently the dissociation of AQP5 from lipid AM, a cell-permeable Ca2+ chelator prevents the cGMP-induced rafts to non-rafts within the APM. The site of action of Ca2+ for increase in AQP5 in the APM. Pretreatment of the parotid tissues the movement and dissociation of AQP5 in parotid gland cells has with ML-9, an MLCK inhibitor, inhibited the ACh-induced not been clarified. In Ca2+-mediated intracellular signal trans- increase in AQP5 in the APM. AQP1 has a cyclic nucleotide 2+ duction, an increase in [Ca ]i has an important role in the binding domain in the C terminus [59]. PKG phosphorylates activation of Ca2+/calmodulin (CaM)-dependent proteins, such as AQP2 on the C terminal residue and increases the insertion of CaM kinase, MLCK, and nitric oxide synthase (NOS). CaM AQP2 into renal epithelial cells [60].ThePKGregulatoryme- kinase is a multifunctional enzyme required for both granule chanism on AQP5 proteins in parotid glands, however, remains mobilization under stimulation conditions and maintenance of unknown. These results are summarized in Fig. 2,suggestingthat secretory capacity under resting conditions in pancreatic β-cells NO/cGMP signal transduction has a crucial role in Ca2+ home- [54]. MLCK regulates capacitative Ca2+ entry [53] and is ostasis in the ACh-stimulated increase in AQP5 in the APM of rat involved in the Ca2+-dependent secretion of insulin [55] and parotid gland. rennin [56]. Nitric oxide (NO) increases cGMP formation through the stimulation of guanylyl cyclase (GC) [57,58]. The possible 7. The mechanisms underlying age- and diabetes-related roles of CaM kinase, NOS, MLCK, and protein kinase G (PKG) impairment of the responsiveness of AQP5 to cholinergic in the regulation of the function of AQP5 were investigated to stimulation clarify the molecular basis of water movement across the biologic membranes in parotid gland cells [25]. Western blot analysis The importance of AQPs in human diseases is recently demonstrated that neuronal (n) NOS is expressed in isolated clarifying [4,5,8–13]. The study of subcellular localization of parotid acinar cells, but endothelial and inducible NOS are not AQP5 provides details of the molecular mechanisms for xeros- [22]. Carboxy-PTIO, an NO scavenger, inhibits ACh- and tomia. Xerostomia is characterized by oral dryness and dif- pilocarpine-induced increases in AQP5 in the APM in rat parotid ficulty performing oral functions and in tolerating dentures. glands. SIN-1 and SNAP, NO donors, mimic the effects of ACh Diabetic patients or aged people often complain of xerostomia, [25]. KT5823 and L-Nil, inhibitors of PKG and NOS, but the mechanisms have not been clarified.

Fig. 2. Schematic representation of signal transduction in M3 muscarinic receptor-induced the translocation of AQP5 in rat parotid glands. M3R; M3 muscarinic receptor, α1R; α1 adrenergic receptors, Gq, G protein that stimulates phospholipase C (PLC); PIP2, phosphatidylinositol 4, 5-bisphosphate; DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate. IP3R, IP3 receptor; Ry3R, type 3; Arg, Arginine; CaM, calmodulin; CaMKII, calmodulin-dependent kinaseII; nNOS, neuronal, nitric-oxide synthase; sGC, soluble guanylate cyclase; PKG, cGMP-dependent protein kinase; cADPr, cADP-ribose; APM, apical plasma membrane; BLM, basolateral plasma membrane. Gray circle is lipid rafts. 1058 Y. Ishikawa et al. / Biochimica et Biophysica Acta 1758 (2006) 1053–1060

The changes in the distribution of AQP5 in rat parotid cells in NOS activity in parotid acinar cells of diabetic rats. Injection of response to M3 agonists have been studied to clarify the mech- insulin to diabetic rats showed the same distribution and anisms underlying age-related xerostomia [62]. ACh and epi- dissociation of AQP5 as those observed in normal control rats nephrine induce increases in AQP5 levels in the APM of parotid [57]. These findings indicate that the decrease in the amount of cells of both young adult and senescent rats. The stimulatory Ca2+ released from intracellular Ca2+ stores via IP3 receptors effect of ACh, but not that of epinephrine, on AQP5 levels in the according to the decrease in the amount of IP3 synthesized APM of the cells decreases markedly during aging. The amounts through agonist-induced activation of M3 mAChRs decreased in of AQP5, M3 mAChRs, inositol trisphosphate (IP3), and Gq/ parotid glands of diabetic rats. Finally, the impairment of 11α proteins do not decrease during aging. This finding in- translocation of AQP5 to the APM in parotid gland cells induced dicates that AQP5 responsiveness to cholinergic, but not by the decline in Ca2+ signaling via M3 mAChRs in parotid adrenergic, stimulation is markedly decreased in the parotid glands is important in the mechanisms underlying diabetes- cells of senescent rats. The changes in NOS activities measured related xerostomia. These findings support also that water is in real time using 4,5-diaminofluorescein/diacetate in isolated secreted from not only acinar cells but also interlobular duct cells parotid acinar cells from young adult and senescent rats treated of rat parotid glands. with ACh or epinephrine coincides with those of the res- ponsiveness of AQP5 in these cells. Confocal images revealed 8. Conclusion that under unstimulated conditions, AQP5 fluorescence locates 2+ in a diffuse pattern in the cytoplasm of parotid acinar cells of The increases in [Ca ]i in salivary gland cells by the both young adult and senescent rats. Ten minutes after the activation of M3 mAChRs and α1-adrenoceptors play a crucial intravenous injection of cevimeline, AQP5 fluorescence does role on salivary fluid secretion. It is documented that water, the not localize in the APM of parotid acinar cells of senescent rats, main component of saliva, is secreted with Na+ and K+ into the in contrast with that of young adult rats [56]. These findings duct system from serous acinar cells in salivary glands as the indicate that the age-related impairment of the responsiveness of primary saliva component. Subsequently, water and K+ are also AQP5 in parotid cells to muscarinic stimulation might account secreted from the interlobular duct cells in the glands and in for the concomitant changes in NOS activity in the cells, and contrast, Na+ is absorbed into the cells as they pass along the might induce age-related xerostomia [62,63]. duct. It was also shown that water was secreted from not only the To study the mechanisms underlying diabetic xerostomia, acinar cells but also the interlobular duct cells of parotid glands changes in the distribution of AQP5 in the interlobular duct cells by the translocation of AQP5 water channel together with lipid of parotid glands were investigated after the administration of rafts to the APM from intracellular vesicles in these cells by Ca2+ cevimeline into the tail vein of control and streptozotocin-in- signaling through M3 mAChRs or α1 adrenoceptors [16]. duced diabetic (diabetic) rats [64]. Confocal images revealed Therefore, the principal sites for water transport are both the that under unstimulated conditions, AQP5 colocalized with acinar cells and interlobular duct cells in the salivary glands. flotillin-2 and GM1, lipid raft markers, with a diffuse pattern in AQP5 is a target molecule for sympathetic or parasympa- the cytoplasm of interlobular duct cells of parotid glands of both thetic control of saliva production. As shown in Fig. 2, the control and diabetic rats. Ten minutes after intravenous injection activation of M3 mAChRs and α1-adrenoceptors induced the of cevimeline, AQP5 levels dramatically increased together with interaction of IP3 receptors and ryanodine receptors with IP3 and flotillin-2 and GM1 in the APM of the cells of control rats, and cADP ribose, respectively, via sGC/PKG signaling. The increase 2+ then AQP5 was again colocalized with flotillin-2 and GM1 in a in [Ca ]i released from intracellular storage sites by the diffuse pattern in the cytoplasm 60 min after the injection. The activation of IP3 receptors and ryanodine receptors induced cevimeline-induced trafficking of AQP5 between the APM and translocation of AQP5 together with lipid rafts from the intracellular structures are not observed in the cells of diabetic intracellular structures to the APM in the interlobular duct rats. Treatment of the parotid tissues with cevimeline induces a cells of rat parotid glands. Then, at the APM, AQP5 located with 2+ decrease in the TritonX-100 insolubility of AQP5 in control rats, lipid rafts was dissociated to non-rafts via the increase in [Ca ]i. but not in diabetic rats. These findings indicate the impairment of Thus, AQP5 in salivary glands [16] and lung [65] is subjected AQP5 trafficking between APM and intracellular structures with to trafficking between the intracellular structures and the APM in lipid rafts and that of AQP5 dissociation from lipid rafts to non- response to neurotransmitters or hormones as well as AQP2 ileal rafts on the APM in the interlobular duct cells of diabetic rat collecting duct cells [8], AQP1 in rat cholangiocytes [66], and parotid glands. The amount of AQP5 does not decrease in AQP8 in hepatocytes [67]. At the APM, AQP5 moves between parotid glands of diabetic rats compared with that of control rats lipid rafts and non-rafts, suggesting that protein–lipid interaction [64]. In contrast, there is a 50% decrease in M3 mAChRs parotid is important to play a role of AQP5. It is very interesting that the glands of diabetic rats. Administration of insulin to diabetic rats impaired responsiveness of AQP5 to M3 mAChRs results in recovers the distribution and dissociation of AQP5 as compared xerostomia. Furthermore, it is important to study the mechan- to control rats. Treatment with cevimeline of isolated parotid isms that control the polarization of AQPs coupled with fluid acinar cells from diabetic rats loaded with DAF-2/DA did not flow and the network of the AQPs involved in fluid secretion induce a fast and significant increase in the fluorescent triazol from salivary glands. Recently, in order to search the domain in formed by DAF-2 and NO in the presence of oxygen as observed AQP5 structures to facilitating to target the APM, much interest in control rats, demonstrating that cevimeline did not stimulate has been focused on N- or C-termini of AQP5 [68]. Further Y. 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